Project Summary To eradicate tuberculosis (TB), the World Health Organization has proposed reducing TB-related deaths from >1 million to just 50,000 per year by 2025. This 95% reduction will not be met by current programs. Thus, the strategic plan to eliminate TB emphasizes the development of new drugs against the causative bacterium, Mycobacterium tuberculosis (Mtb). The Mtb cell envelope has thus been the target of many efforts to find novel anti-TB therapeutics. However, discovery and validation of new targets is hampered by our limited understanding how the cell envelope is made. Enzymes that make mycomembrane lipids have been characterized, but the mechanism of subsequent lipid incorporation into the mycomembrane remains poorly defined. We and others have identified a pathway that is required for both the transport and the synthesis of diverse mycomembrane lipids. Based on our published work and preliminary data, we hypothesize that the lipoprotein LprG and membrane protein Rv1410c play a broad role in mycomembrane biogenesis by mediating a crucial step in transporting lipids beyond the cytoplasmic membrane and into the mycomembrane. To test this model, we will pursue the following aims: (1) Determine how LprG and Rv1410c regulate lipid transport to the mycomembrane and (2) Determine how LprG-Rv1410c regulates the addition of mycolic acid-bearing virulence factors to the mycomembrane. Our innovation is to test a novel model for mycomembrane biogenesis and to do so we will leverage our expertise in mycobacteriology and lipid biochemistry to achieve our long-term goal of understanding lipid transport processes that enable the mycomembrane to form and contribute to virulence. The successful completion of these Specific Aims will provide important advances in our understanding of a virulence-associated pathway that mediates these processes in the Mtb cell envelope.